The tardigrade-derived mitochondrial abundant heat soluble protein improves adipose-derived stem cell survival against representative stressors

Human adipose-derived stem cell (ASC) grafts have emerged as a powerful tool in regenerative medicine. However, ASC therapeutic potential is hindered by stressors throughout their use. Here we demonstrate the transgenic expression of the tardigrade-derived mitochondrial abundant heat soluble (MAHS) protein for improved ASC resistance to metabolic, mitochondrial, and injection shear stress. In vitro, MAHS-expressing ASCs demonstrate up to 61% increased cell survival following 72 h of incubation in phosphate buffered saline containing 20% media. Following up to 3.5% DMSO exposure for up to 72 h, a 14–49% increase in MAHS-expressing ASC survival was observed. Further, MAHS expression in ASCs is associated with up to 39% improved cell viability following injection through clinically relevant 27-, 32-, and 34-gauge needles. Our results reveal that MAHS expression in ASCs supports survival in response to a variety of common stressors associated with regenerative therapies, thereby motivating further investigation into MAHS as an agent for stem cell stress resistance. However, differentiation capacity in MAHS-expressing ASCs appears to be skewed in favor of osteogenesis over adipogenesis. Specifically, activity of the early bone formation marker alkaline phosphatase is increased by 74% in MAHS-expressing ASCs following 14 days in osteogenic media. Conversely, positive area of the neutral lipid droplet marker BODIPY is decreased by up to 10% in MAHS-transgenic ASCs following 14 days in adipogenic media. Interestingly, media supplementation with up to 40 mM glucose is sufficient to restore adipogenic differentiation within 14 days, prompting further analysis of mechanisms underlying interference between MAHS and differentiation processes.


Mitochondrial localization and morphology analysis
AcGFP1-and MAHS-transgenic ASCs were seeded at an initial density of 10,000 cells/well in routine culture media.After 24 h mitochondrial localization and morphology was assessed.Four sample replicates of each genotype were obtained.Samples were stained with 125 nM MitoView Fix 640 (Biotium #70082, Fremont, CA) in cell culture media.After 2 h, samples were fixed for 5 min with 4% paraformaldehyde (ThermoFisher #T353500, Riverside, CA) in PBS and subsequently permeabilized for 5 min with 0.25% Triton X-100 (ThermoFisher #BP151500, Riverside, CA) in PBS.Samples were incubated overnight at 4 °C in a stain solution of [1:750]  DyLight Phalloidin 554 (Cell Signaling Technology #13054, Danvers, MA) in 1× DAPI (Invitrogen #D1306, Waltham, MA).Samples were washed three times with PBS and stored in PBS at 4 °C before imaging.All samples were imaged using a DFC9000 GT sCMOS camera on a DMi8 inverted microscope (Leica Microsystems, Chicago, IL).A 64-position tile scan of ~ 0.95 cm 2 area was obtained for each sample with 1 µm steps to focus each image.Four separate cell platings were used as biological replicates.
A custom MATLAB script (MATLAB R2022a; MathWorks, Natick, MA) was written to quantify the Pearson correlation coefficient between expressed protein localization (AcGFP1 or MAHS) and mitochondrial morphology (MitoView).For all analysis, quantification of each experimental group was obtained from averaged cell counts of 64 positions per sample.Mitochondrial morphology characteristics were quantified using the MiNA software 66 .Pairwise comparison between Pearson correlation coefficients and mitochondrial morphology characteristics of AcGFP1-and MAHS-expressing ASCs were performed using two-sample t-tests with significance of α = 0.05 (MATLAB).Medians and individual data points are indicated by black bars and triangles, respectively.The dataset utilized for generating all graphical representations and conducting statistical analyses is available in the supplementary materials.

Media depletion tolerance
Experiments were performed in 48-well plates.AcGFP1-and MAHS-expressing ASCs were seeded at an initial density of 10,000 cells/well in routine culture media.After 24 h, culture media was replaced with media diluted with various concentrations of DPBS-no further media changes were performed following initial administration.DPBS concentrations evaluated in ASC52telos over a period of 72 h include 0%, 70%, 80%, and 90% DPBS.Cells were fixed as described above, incubated in DAPI and [1:750] DyLight Phalloidin 554 solution and imaged as described above.Three separate cell platings were used as biological replicates.Viability analysis was also performed using viability/cytotoxicity assay kit (#30002; Biotium, Fremont, CA) solutions diluted to final concentrations of Calcein-AM [2 µM] and EthD-III [4 µM] in PBS.However, dead cell loss due to cell detachment over the extended culture time resulted in high live/dead ratios not representative of the cell loss (Figure S1B).Consequently, the cell population remaining attached was considered as the primary metric of media depletion tolerance.
A custom MATLAB script was written to quantify DAPI counts within phalloidin cell borders and express outputs as a percentage of cell viability normalized to respective control AcGFP1 or MAHS samples.For all analysis, quantification of each experimental group was obtained from averaged cell counts of 64 positions per sample.Analysis of the extent to which the relationship between DPBS treatment and cell population varied with genotype was performed with N-way analysis of variance (ANOVAN).Post hoc comparisons at each tested DPBS concentration were performed using Tukey's honestly significant difference (Tukey's HSD) test.All statistical tests were performed in MATLAB.Statistical tests and data visualization were performed in MATLAB as described above.The dataset utilized for generating all graphical representations and conducting statistical analyses is available in the supplementary materials.

Freezing tolerance
AcGFP1-and MAHS-expressing ASCs were routinely passaged, resuspended in 100% bovine calf serum (BCS, Fisher Scientific #SH3007303HI, Hampton, NH) and deposited directly into liquid nitrogen (flash freezing) or in a cell freezing container for controlled freezing (− 1 °C/min) down to − 80 °C.After 7 days, cryovials were removed from liquid nitrogen storage, centrifuged, and resuspended in viability/cytotoxicity assay kit (#30002; Biotium, Fremont, CA) solutions diluted to final concentrations of Calcein-AM [2 µM] and EthD-III [4 µM] in PBS.Samples were transferred into 24 well plates and incubated for 30 min at room temperature.Cells were imaged as described above.Three separate cell suspensions were used as biological replicates.
A custom MATLAB script was written to quantify cell viability.For all analysis, quantification of each experimental group was obtained from averaged cell counts of 36 positions per sample.Pairwise comparisons at each tested cryopreservation period were performed using two sample t-tests.All statistical tests were performed in MATLAB.Statistical tests and data visualization were performed in MATLAB as described above.The dataset

DMSO tolerance
Experiments were performed in 48-well plates.AcGFP1-and MAHS-expressing ASCs were seeded at an initial density of 10,000 cells/well in routine culture media.After 24 h the cells were treated with varying concentrations of dimethyl sulfoxide (DMSO; Gaylord Chemical Company #MFCD00002089, Covington, LA).Cells were treated for 72 h with 0%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 5% DMSO.Cells were incubated in DAPI and [1:750] DyLight Phalloidin 554 solution and imaged as described above.Three separate cell platings were used as biological replicates.Viability analysis was also performed using viability/cytotoxicity assay kit (#30002; Biotium, Fremont, CA) solutions diluted to final concentrations of Calcein-AM [2 µM] and EthD-III [4 µM] in PBS.Similar to media depletion tolerance testing, detachment of dead cells led to live/dead ratios not representative of cell loss (Figure S2B).Consequently, cell population was considered as the primary metric of DMSO tolerance.
The custom MATLAB script was used to quantify DAPI counts and calculate relative cell population as described above.Analysis of the extent to which the relationship between DMSO treatment and cell viability varied with genotype was performed with analysis of covariance (ANCOVA) and Tukey's honestly significant difference (Tukey's HSD) test were conducted as described above.The dataset utilized for generating all graphical representations and conducting statistical analyses is available in the supplementary materials.

Biomaterial fabrication
9 g of Sylgard 184 silicone elastomer base and 1 g of silicone curing agent (WPI SYLG184, Sarasota, FL) were vigorously mixed and placed in a desiccator vacuum chamber for 30 min to remove air bubbles.The solution was then poured into a 90 mm petri dish and cured in an oven at 60 °C for 2 days to prevent upstream solvent leaching into cell culture.¼" diameter circular holes were cut into cured polydimethylsiloxane (PDMS), and PDMS "barriers" were firmly pressed onto 22 × 22 mm glass coverslips.PDMS barriers and coverslips were sterilized by autoclave and utilized for injection tolerance experiments.

Injection tolerance
Prior to injection, AcGFP1-and MAHS-transgenic ASCs were washed three times with DPBS (+ , +), routinely passaged and resuspended at a density of 5000 cells/µL in viability/cytotoxicity assay kit (Biotium #30002, Fremont, CA) solutions diluted to final concentrations of Calcein-AM [2 µM] and EthD-III [4 µM] in PBS.The resuspended solutions were immediately injected onto glass coverslips surrounded by PDMS barriers at an injection rate of 1,000 µL/min.Samples were imaged on an inverted microscope as described above.A 36-position tile scan of ~ 0.53 cm 2 area was obtained for each sample with 1 µm steps to focus each image.Six cell platings were used as biological replicates for each experimental group.
The custom MATLAB script was used to calculate percentage of Calcein-AM-expressing cells as described above.Analysis of the extent to which the relationship between needle gauge and cell viability varied with genotype was performed through 2-way analysis of variance (2-way ANOVA) and Tukey's honestly significant difference (Tukey's HSD) test as described above.The dataset utilized for generating all graphical representations and conducting statistical analyses is available in the supplementary materials.

Adipogenic differentiation
Experiments were performed in 24-well plates.AcGFP1-and MAHS-expressing ASCs were seeded at an initial density of 10,000 cells/well in routine culture media.After 24 h, cells were treated with adipogenic media for 7, 14, and 21 days with media changes every 3 days.Adipogenic media components include DMEM/F-12 50 A custom MATLAB script was written to quantify adipogenic expression in terms of percent of cellular BODIPY positive area.For all analysis, quantification of each experimental group was obtained from averaged cell counts of 64 positions per sample.Analysis of the extent to which the relationship between culture period and BODIPY positive area varied with genotype was performed through was performed through N-way analysis of variance (ANOVAN) and Tukey's honestly significant difference (Tukey's HSD) test as described above.The dataset utilized for generating all graphical representations and conducting statistical analyses is available in the supplementary materials.

Osteogenic differentiation
Experiments were performed in 24-well plates.AcGFP1-and MAHS-expressing ASCs were seeded at an initial density of 10,000 cells/well in routine culture media.After 48 h cells were treated with osteogenic media (osteocyte differentiation tool; ATCC PCS-500-052, Manassas, VA).Media was exchanged every 4 days until the culture endpoint.Samples were fixed for 5 min with 4% paraformaldehyde and incubated in the dark for 30 min at room temperature in a stain solution of naphthol AS-MX phosphate (0.1%, research organics #1596-56-1, Cleveland, OH) and fast red violet LB salt (0.1%, electron microscopy sciences #32348-81-5, Hatfield, PA) in 56 mM AMPD buffer, pH 9.9 (2-amino-2-methyl-1,3-propanediol, TCI America #A0332, Portland, OR).Samples were washed three times with PBS and stored in PBS at 4 °C before imaging.Representative samples were captured with a brightfield microscope with MU300 digital camera (AmScope, Irvine, CA).Three separate cell platings were used as biological replicates.
A custom MATLAB script was adapted from a published color deconvolution script to quantify osteogenic expression in terms of the mean pixel intensity (MPI) of cellular alkaline phosphatase activity 67,68 .For all analysis, quantification of each experimental group was obtained from averaged MPIs of six representative brightfield images per sample.Pairwise comparisons at each tested culture period were performed using two-sample t-tests.All statistical tests were performed in MATLAB as described above.The dataset utilized for generating all graphical representations and conducting statistical analyses is available in the supplementary materials.

MAHS expression in ASCs is localized to the mitochondria
We first assessed the localization of MAHS expressed in ASCs.Fluorescent localization allows for confirmation of the expression of MAHS in ASCs (Fig. 1).Qualitatively, AcGFP1-tagged MAHS was observed to localize to the mitochondria in ASCs, while AcGFP1 alone (control) was expressed nonspecifically throughout the cytosol.Quantitatively, MAHS expression and MitoView had a significantly larger Pearson correlation coefficient than AcGFP1 and MitoView.These findings are consistent with transient expression studies of MAHS in human HEp-2 and HEK293T cells 60 .We did not detect any substantial difference in mitochondrial morphology between cell genotypes, using the mitochondrial network analysis (MiNA) software to assess several morphometric parameters (Figure S3A-K) 66 .We also did not detect any change in MitoView intensity in MAHS-transgenic ASCs(Figure S3L).

MAHS confers DMSO tolerance to ASCs
To assess the effect of MAHS expression on DMSO tolerance, ASCs were incubated in culture media containing various concentrations of DMSO for 72 h.Cell population after DMSO treatment, as measured by cell count normalized to control (0%) treatment across 1-5% treatment doses, was significantly higher in MAHS-transgenic ASCs than in AcGFP1-transgenic ASCs (ANCOVA, p = 0.0033).Further, survival was increased in MAHSexpressing ASCs in media with 1% (49% ± 11%), 2% (45% ± 8.8%), 3% (23% ± 5.4%), and 3.5% (14% ± 5.2%) DMSO (Fig. 2).These data suggest a MAHS-influenced tolerance to chronic exposure to low amounts of DMSO.We also tested live-dead viability under these treatment conditions (Figure S2); however, cell population was a more appropriate stress metric due to artificial inflation of live cell percentages resulting from cell loss over the 72 h treatment period.Relatedly, we tested viability after cryopreservation without DMSO using both slow and flash freezing.However, MAHS did not offer improved survival in this context (Figure S4).

MAHS confers metabolic stress tolerance to ASCs
To analyze the effect of MAHS expression on ASC population in response to prolonged nutrient depletion, ASCs were incubated in culture media diluted to 30%, 20%, or 10% with DPBS containing calcium and magnesium for 72 h and assessed via cell counts compared to full media controls (Fig. 4).There was a significant protective effect of MAHS expression against media depletion groups (ANOVAN, p = 2.0e-9).Further, cell population was significantly higher in MAHS-transgenic ASCs than in AcGFP1-transgenic ASCs in 30% (61% ± 8.6%) and 20% (23% ± 1.0%) media (α < 0.05, Tukey's HSD).These data suggest that MAHS expression aids in metabolic stress resistance in ASCs.We also tested live-dead viability under these treatment conditions (Figure S1); however, as previously mentioned, substantial cell detachment over the 72 h treatment period skewed live cell measurements, and cell population was instead considered as the primary stress metric.

MAHS-transgenic ASCs preferentially differentiate down osteogenic over adipogenic lineages
Stem cell therapies may rely on differentiation into specific cell types; this may be influenced by the expression of an exogenous protein.To test the effect of MAHS expression on ASC differentiation, we used standard methods to induce either adipogenic or osteogenic differentiation 70 .Osteogenic differentiation, as measured by quantification of the early osteogenic marker alkaline phosphatase (ALP) activity, was significantly higher in MAHS-expressing ASCs than in AcGFP1-expressing ASCs following a 14 day culture period (73.7510% ± 6.5310%) in osteogenic media (Fig. 5).Conversely, adipogenic differentiation, as measured by quantification of the neutral lipid droplet marker BODIPY, was significantly higher in AcGFP1-expressing ASCs than in MAHS-expressing ASCs following 7 day (3.644% ± 0.5228%) and 14 day (9.568% ± 0.5199%) culture periods in adipogenic media (Fig. 6).These data suggest that MAHS expression affects ASC differentiation in favor of an osteogenic lineage over an adipogenic lineage.

Transgene localization to mitochondria
In tardigrades, the MAHS protein plays a critical role in guarding against desiccation-related mitochondrial damage, and its protective activity against metabolic stress associated with hyperosmotic conditions is documented in HEp-2 cells 60 .However, potential for application of MAHS expression in mammalian cells for therapeutic purposes is currently limited due to the lack of knowledge of whether MAHS performs similar protective functions in therapeutic cells such as ASCs.Here, we demonstrated that the AcGFP1-tagged MAHS protein is stably expressed in ASCs and is localized to the mitochondria (Fig. 1).This is consistent with other studies of in vitro expression of MAHS in mammalian cells 60 .Although direct experimental data is currently lacking, it has been hypothesized that MAHS may stabilize the two mitochondrial phospholipid bilayers by maintaining appropriate spacing between polar head groups of membrane phospholipids 58 , this may be part of the protective mechanism in ASCs.As an intrinsically disordered protein, MAHS may undergo multiple conformational changes to secondary structure to confer multistress tolerance, similar to the PvLEA-22 peptide, which transitions from an alpha-helical structure to β-sheet conformation upon contact with cell membrane structures 58,[72][73][74][75] .It is important to note that MitoView 640 staining intensity is independent of mitochondrial membrane potential, unlike some varieties of mitochondrial dyes 76,77 .Therefore, while we did not observe morphological changes to mitochondria, it is possible that mitochondrial membrane potential may be altered in MAHS-expressing ASCs.If so, alteration of membrane potential may contribute to the restrained adipogenic differentiation capacity observed in

DMSO tolerance
DMSO is a valuable mammalian cell cryoprotectant: it is commonly used to protect ASCs and other therapeutic cell types from cell membrane damage caused by intracellular ice formation during flash freezing, as well as dehydration and hyperosmotic stress due to solute concentration and solvent freezing during slow cooling 18,19 .However, prolonged exposure to DMSO compromises mitochondrial membrane integrity through generation of damaging ROS species and lowered mitochondrial membrane potential, and facilitates release of calcium from the endoplasmic reticulum to initiate apoptosis [22][23][24][25][26] .In addition to limiting viability of stem cells for therapeutic purposes, patients receiving injections or grafts containing these cells have been documented to experience severe DMSO-specific neurological and immunological responses [27][28][29][30] .Chronic DMSO exposure studies show that the MAHS protein provides protection to ASCs against cell damage induced by up to 3.5% DMSO (Fig. 2).Given the mitochondrial localization of MAHS and hypothesized membrane interaction 58 , it is possible that MAHS supports mitochondrial membrane integrity and reduces DMSO-induced membrane permeabilization.However, MAHS expression did not protect ASCs from stressors associated with freezing without the use of a cryoprotectant; this is contrary to the tolerance exhibited by tardigrades during freezing (Figure S4) 60,78 .

Injection tolerance
Injection of ASCs is a treatment method for diseased, damaged, and nutrient deprived tissues.Clinical needle diameters are often reduced for patient comfort and to permit safe injection into sensitive tissues 31,[36][37][38] .However, cell injections expose cells to high shear and extrusion stresses, resulting in viability loss.It has been reported for cell viability loss rates to be as high as 68-99% following therapeutic injection into target tissues [39][40][41] .Injection studies across a range of clinical needle gauges (i.e., 18-34 gauge) indicate that MAHS expression provides protection to ASCs against shear stress (Fig. 3).While the specific mechanism is out of the scope of this work, there are several potential mechanisms for this effect.In other cell types, it has been shown that fluid shear stress can directly rupture cell membranes, stimulate the release of intracellular calcium from the endoplasmic reticulum and cytochrome c from the mitochondria to initiate apoptotic signaling, and promote expression of additional apoptosis regulating genes 60,[79][80][81] .Further, it has also been shown that damaging ROS species produced in mitochondria are implicated as secondary messengers in the modulation of shear-induced gene expression in endothelial cells 82 .As previously speculated, MAHS may be directly preventing mitochondrial membrane disruption 58 .MAHS may also indirectly strengthen the cell membrane or cortical cytoskeleton.For example, prior work has shown increased stiffness in multiple stem cell types prior to and after osteogenic differentiation 83,84 ; given the propensity for osteogenic differentiation in MAHS-expressing ASCs, it is possible that these cells innately possess stiffened membranes and matrices that are resistant to deformation.

Metabolic stress tolerance
Stem cell therapies may be injected into ischemic tissues for repair and regeneration of damaged tissue, where metabolic stress is common in response to limited oxygen and nutrient supply caused by impaired perfusion 85,86 .Specifically, starvation is known to induce oxidative stress through ROS production, decreased mitochondrial membrane potential, and activation of the apoptosis cascade through calcium release from the ER [20][21][22][23][24] .Here, the MAHS protein provides protection to ASCs against metabolic stress induced by nutrient depletion, suggesting potential for increased viability in damaged or ischemic engraftment sites (Fig. 4).While the specific mechanism is outside the scope of this study, potential mechanisms may include reduced energy generation or ROS tolerance.While not quantified in this study, reduced mitochondrial potential caused by MAHS expression could potentially indicate lower overall metabolic activity 87 .However, this is speculative, given that no prior studies of MAHS-influenced changes to mitochondrial potential have been performed.Mitochondria also contain ROSneutralizing enzymes to counteract oxidative stress, including manganese superoxide dismutase, and the GPx4 isoform of glutathione peroxidase [88][89][90][91][92][93] , and expression of such enzymes may be upregulated in MAHS-expressing ASCs.Prior studies in HEp-2 cells also showed that transient MAHS expression was protective to hyperosmotic stress; a similar mechanism could be contributing here 60 .

Differentiation
ASCs are partially characterized by their multilineage differentiation capacity: some common differentiation lineages for therapeutic applications include adipogenic and osteogenic cell types 94,95 .This allows for potential application of ASCs to treat a variety of conditions, including bone fractures, osteoarthritis and osteoporosis, spinal fusions, craniofacial and soft tissue defects, and cosmetic defects requiring enhanced adipogenic tissue volume 70,[96][97][98][99][100][101][102] .Overall, the multilineage differentiation capacity in ASCs is a key contributor to their therapeutic potential.However, the presented studies in MAHS-expressing ASCs suggest that differentiation may be skewed in favor of osteogenic lineages.Neutral lipid droplet staining of MAHS-transgenic ASCs cultured in adipogenic media revealed a lower lipid count relative to ASCs expressing only AcGFP1, suggesting interference of the MAHS protein in ASC adipogenesis or lipogenesis (Fig. 5).Further, alkaline phosphatase staining of MAHStransgenic ASCs cultured in osteogenic media demonstrated a higher level of alkaline phosphatase activity than ASCs expressing only AcGFP1 (Fig. 6).
While mechanism is not explored herein, one potentially compelling explanatory mechanism is hyperactivation of the AMPK/FOXO signaling pathway by MAHS through high basal AMPK concentrations.Expression of MAHS, an exogenous protein, may affect ATP generation in the mitochondria.Metabolic deficits can subsequently influence ASC differentiation processes through a number of regulators.Low glucose conditions activate both AMPK and FOXO to promote osteogenic over adipogenic differentiation 71 .Indeed, the restoration of adipogenic differentiation capacity in MAHS-ASCs following administration of excess glucose, a known AMPK inhibitor, to ASC cultures provides support for future investigation of AMPK as an effector of MAHS activity in ASCs (Figs. 7 and 8).

Limitations and future work
Though MAHS expression in ASCs demonstrates significant potential for improved stress tolerance, several limiting factors must be considered.MAHS-transgenic ASCs are significantly less resistant to freezing, which is in contrast to the increased resistance of tardigrades to freezing (Figure S4) 60,78 .Additionally, MAHS-transgenic ASCs have only been analyzed in the context of constitutive expression; other expression modalities such as mRNA transfection may be more relevant to therapeutic applications 103 .Further, MAHS expression appears to push ASCs down an osteogenic lineage, which may be detrimental to therapeutic applications where non-osteogenic cell types are desired.However, bone and osteoarticular therapies may particularly benefit from targeted use of MAHS-expressing ASCs; this will be considered in future studies.Finally, there may be safety considerations associated with intrinsically disordered proteins, which should be assessed specifically for MAHS.As an example, amyloid-β peptide can form insoluble fibrillar protein aggregates, which have been linked to amyloidogenicassociated diseases, although specific mechanisms remain poorly understood [104][105][106] .Notably, there is no existing data describing the effect of MAHS expression on immunogenicity or its efficacy in vivo, although studies on other intrinsically disordered proteins indicate this may not be a concern 107,108 .Future studies will evaluate the viability and safety of MAHS expression in vivo, while mechanistic studies will be conducted on the molecular mechanisms downstream of MAHS that result in the functional changes observed.

Figure 1 .
Figure 1.Mitochondrial localization of MAHS.(A) Quantification of Pearson correlation coefficient for the overlap between expressed protein localization and mitochondrial morphology (two-sample t-test, n = 4).The denoted p-value indicates the pairwise difference in GFP/MitoView correlation between the genotypes.(B) Maximum projections of the overlap between expressed protein localization (AcGFP1 or AcGFP1-MAHS, cyan) and mitochondrial morphology (MitoView, yellow) in ASC52telos transduced with either the AcGFP1 or AcGFP1-MAHS transgene.Scale bars are 25 µm.

Figure 2 .
Figure 2. DMSO stress.(A) Maximum projections of the nuclear (DAPI, cyan) and cytoskeletal (phalloidin, magenta) structures of ASC52telos transduced with either the AcGFP1 or MAHS transgene following 72 h of DMSO treatment.Scale bars are 200 µm.(B) Quantification of cell population in AcGFP1-and MAHSexpressing ASCs following a 72 h DMSO treatment (ANCOVA, Tukey's HSD, n = 3).Cell population quantification was normalized to cells cultured without DMSO for 72 h.Denoted p-values indicate pairwise difference in cell population between the genotypes at each specific DMSO concentration.

Figure 4 .
Figure 4. Metabolic stress.(A) Maximum projections of the nuclear (DAPI, cyan) and cytoskeletal (phalloidin, magenta) structures of ASC52telos transduced with either the AcGFP1 or MAHS transgene following 72 h of media depletion.Scale bars are 300 µm.(B) Quantification of cell population in AcGFP1-and MAHSexpressing ASCs following a 72 h media depletion (ANOVAN, Tukey's HSD, n = 3).Cell survival quantification was normalized to cells cultured in 100% media for 72 h.Denoted p-values indicate pairwise difference in cell population between the genotypes at each specific media concentration.